The overall objective of our research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to focus on evolutionarily conserved chromatin remodeling enzymes that facilitate transcription by remodeling chromosome structure. During the next budget period, we will focus on the yeast SWR-C remodeling enzyme that is dedicated to the ATP-dependent deposition of the H2A.Z histone variant within nucleosomes that flank promoters of genes transcribed by RNA polymerase II, as well as nucleosomes that flank chromatin boundary elements, centromeres, and replication origins. Mammalian homologs of SWR-C, including the p400/Tip60 complex, also control H2A.Z deposition and they are key for proper stem cell function, genome stability, development, and gene expression. During the past budget period, we identified a novel regulatory interaction between SWR-C and the acetylation of lysine 56 of histone H3 (H3-K56Ac) that regulates nucleosome dynamics, noncoding RNA expression, and assembly of large-scale, chromosome interaction domains (CIDs) that are related to mammalian topologically-associated domains (TADs). Our overall strategy is to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the roles of SWR-C and H3- K56Ac in vivo and the biochemical mechanisms by which these chromatin regulators alter nucleosome structure in vitro. Experiments described in this proposal address three aims. The first aim will dissect the biochemical mechanism of H2A.Z deposition and its regulation by H3-K56Ac. Here we will employ quantitative, fluorescence-based assays to define steps of the histone dimer exchange reaction. Hydroxyl-radical footprinting and site-specific histone-DNA crosslinking approaches will also be employed to define how SWR-C binds and alters nucleosomal DNA during the remodeling reaction. We will dissect the role of different subunit modules in this reaction, focusing initially on the conserved Swc5 and Rvb1/Rvb2 subunits. We will also exploit state-of-the-art, protein crosslinking/mass spectrometry methods to explore the subunit topology of SWR-C and to probe how SWR-C subunits interact with different nucleosomal substrates, with emphasis on understanding how histone H3-K56Ac alters the substrate specificity of the dimer exchange reaction. Aim 2 proposes a combination of nascent transcript sequencing (Net-seq), Transcript leader sequencing (TL-seq), and DNA tiling arrays to probe interactions between H2A.Z, H3-K56Ac, and the RNA exosome in the regulation of the yeast transcriptome. We also test the hypothesis that genome-wide hyperacetylation of H3-K56Ac leads to aberrant ncRNA expression that causes genomic instability. Studies described in Aim 3 investigate how CIDs are established and maintained. This aim will employ Micro-C analyses to test whether the role of H3- K56Ac in CID structure is due to replication-coupled nucleosome assembly, and we test whether the requirement for SWR-C (H2A.Z) is linked to changes in transcription within CIDs. Finally, we use genome- wide, in vitro reconstitution of promoter nucleosome architecture to test whether formation of nucleosome depleted regions (NDRs) is sufficient to direct assembly of CIDs. OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page Continuation Format Page